This project will focus on expanding the therapeutic range of HSV-1 vectors for treatment of brain tumors and on creating zones of resistance to tumor growth in the normal brain by developing novel vectors and delivery modalities. The current proposal plans to increase the therapeutic capacity of the oncolytic HSV-1 vector, MGH2 in two ways: by enhancing infection of glioblastoma (GBM) cells through targeting surface antigens by virions;and by combining oncolytic vectors with amplifying/integrating HSV/AAV amplicon vectors to increase and sustain therapeutic transgene levels. We will also attempt to create a zone of resistance to tumor growth in normal brain via AAV vectors and neuroprecursor cells (MFCs) expressing secretable therapeutic proteins. NPCs and chimeric immune receptor (CIR) lymphocytes will be used to target invasive tumor cells. All studies will be carried out in culture and in intracranial human glioma models in nude mice using bioluminescence imaging reporters to track vector and protein delivery, cell fate and tumor growth. An immunocompetent syngeneic rat GBM model will also be used in evaluating CIR lymphocytes. Aim 1 will explore selective infection of glioblastoma cells expressing the mutant EGF receptor, EGFRvIII, by modification of the glycoprotein C envelope protein of HSV-1 virions for presentation of antibodies to this receptor. Aim 2 will evaluate the ability of CIR lymphocytes to target to EGFRvIII and VEGFR receptors in tumor foci. In Aim 3, AAV vectors and genetically modified NPCs will be used to deliver secretable forms of the tumorspecific apoptotic protein, TRAIL, and the anti-angiogenic factor, Flkl to normal brain to create a region of resistance to tumor growth. Aim 4 will employ a dual gene delivery system combining hybrid amplicon vectors incorporating p5 and ITR DNA elements from AAV, and Rep 78/68 proteins as fusions with the HSV-1 virion tegument protein, VP16. Co-infection with oncolytic HSV-1 or a subset of HSV-1 genes will be evaluated for replicative amplification and genomic integration of amplicon-encoded transgenes. This project will use expertise on oncolytic virus (Project 1, Chiocca), in vivo imaging (Project 3, Weissleder), biostatistics (Core A, Finkelstein), HSV vector stocks (Core B, Krisky/Glorioso), and human GBM cells and neuropathology (Core C, Louis). These studies are designed to complement and increase the therapeutic impact of the HSV-1 oncolytic vector strategy for cancer treatment and to explore the possibility of creating regions of the brain resistant to tumor growth by sustained release of therapeutic proteins, using means deemed compatible with human trials.